The project has addressed the following areas in the past year: 1. Association of highly charged intrinsically disordered proteins. In collaboration with Ben Schuler (University of Zurich) and Birthe Kragelund (University of Copenhagen), we have been studying the formation of a complex between histone H1 and the protein prothymosin alpha, believed to act as a chaperone for H1. Experimentally, it was found that the complex has extremely high (picomolar) affinity, but with no evidence for formation of structure on binding. Using a minimalist coarse-grained model, we were able to reproduce the experimental FRET data accurately and to explain the NMR data. Our simulations show the key role of electrostatics in determining the structural ensemble of the bound complex. (Ref. 1). We are currently expanding this work to address the kinetics of association and substitution for these complexes, as well as to address possible coacervation at higher protein concentrations. (R. Best) 2. Characterization of Alzheimer's Abeta monomer. The peptides Abeta40 and Abeta42 constitute the majority of the plaques found in the brains of Alzheimers patients. Understanding the mechanism of assembly of these plaques formed by fibrils of the Abeta peptides is therefore a major research objective. We have collaborated with the group of Hoi-Sung Chung (LCP, NIDDK) to study structure formation in Abeta40/42 monomers. Both experiment and simulation are consistent with a completely unstructured monomer, in contrast to previous work suggesting significant secondary structure population in the monomer. (Ref 2). We are currently working on studying the mechanism of secondary nucleation via enhanced sampling (manuscript under review), and we have used an allocation of time on the ANTON supercomputer to look at formation of oligomeric Abeta42 species (M. Bellaiche, R. Best) 3. Folding of membrane proteins (Collaboration with Mark Sansom, University of Oxford). We have used all-atom simulations to examine the assembly of the glycophorin dimer, a prototype for transmembrane protein folding. While the best current force field correctly predicts the structure of the native dimer, it is extremely unstable a fact that was not appreciated previously due to the very extensive sampling needed for this (Ref 3). We used experimental data to modify the force field to obtain a more realistic dissociation constant. We have used the modified force field to study the kinetics and mechanism of TM helix association via transition-path sampling (manuscript in preparation). (J. Domanski, R. Best) 4. Protein sequence evolution and design. Recent work has shown the potential of models parameterized on sequence alignments to capture correlations between residues in folded proteins. Such models intrinsically are able to assign a statistical energy to any sequence for its ability to fold into the structure corresponding to the sequence alignment (Ref 4). Taken to its logical extreme, this type of model could be used to design novel protein sequences, and we have tried this also, designing sequences which fold to each of the GA and GB domains of staphyloccocal protein G, as well as to an SH3 domain. We have now obtained well-folded examples of novel sequences folding to each of these domains by this approach, tested experimentally by John Louis (LCP, NIDDK), and have determined NMR structures for representative examples of these proteins with James Baber (LCP, NIDDK) (Ref 5). In the final thread of the project, we are trying to design sequences which fold into different structures using the statistical models, thus formalizing an idea which has been pioneered empirically by Philip Bryan (University of Maryland). (P. Tian, R. Best) 5. Co-translational protein folding of titin. We are using coarse-grained simulations to interpret experiments in the groups of Jane Clarke (Cambridge University), Gunnar von Heijne (Stockholm University) and Susan Marqusee (Berkeley) on co-translational folding on the ribosome. The experiments use an arrest peptide to stop translation by becoming stuck in the ribosome exit tunnel. However, there is a spontaneous rate of escape from the arrest, which may be increased by any force exerted by the protein. The yield of protein escaping the ribosome after a given incubation time is used as a measure of the force exerted. Using the forces measured in simulations, and experimental data on the force-dependence of the escape rate from earlier experiments, we have developed a kinetic model that allows us to compare directly with the yield of protein obtained in the experiment. The maximum yield is a trade-off between the maximum force exerted by the folded state (early in translation) and the population of the folded state (highest late in translation). (2 manuscripts under review) (P. Tian, R. Best) 6. Development of coarse-grained models for protein phase separation (collaboration with Jeetain Mittal, Lehigh University). Recent work has shown that so-called stress granules formed in cells as a result of stress are a protein-rich phase which can be reconstituted in vitro from selected purified components. We have developed a coarse-grained model which can capture the sequence-dependence of the phase behaviour and the intermolecular interactions stabilizing the high density phase. Together with special simulation methods, we can determine phase diagrams from the model (Ref 6). We have now used this model to related the critical temperature for phase separation to properties of the protein monomers and dimers. This should allow prediction of phase separation properties from studying smaller, simpler systems in simulation or experiment (Ref. 7) (W. Zheng, R. Best) 7. Methods for interpreting experiments on intrinsically disordered proteins. In 2016, we resolved a controversy in interpretation of FRET and SAXS experiments on intrinsically disordered proteins, showing that it arose from oversimplified analysis methods. However, the methods we used to interpret the experiments were too burdensome for general use. We have therefore developed methods for interpreting FRET (Ref 8) and SAXS (Ref. 9) experiments on intrinsically disordered proteins which are both straightforward to use and also give accurate results, unlike earlier methods. We recently rebutted a claim that the results of SAXS and FRET experiments are in contradiction (Ref 10). (W. Zheng, R. Best) 8. Instrumental effects on folding kinetics studied by single-molecule atomic force microscopy (AFM). In collaboration with David de Sancho, we have shown that the slow protein folding kinetics observed when folding is monitored by AFM are a consequence of the slow response of the instrument. Nonetheless, the experiments still yield useful information on the folding (Ref. 11). 9. Interpretation folding psi-values. Psi-values have been proposed as an alternative to phi-values, in which a pair of histidine residues is engineered into a protein in order to create a divalent binding site for metal ions such as Zinc. However, several experimentalists have raised questions over the interpretation of psi-values. An additional concern is the time scale of ion association/dissociation relative to folding, since the time scale for ion binding is comparable to that of protein folding transition paths. We are using coarse-grained simulations to investigate under what experimental conditions psi values might be easily interpreted in terms of folding mechanism. (W. Li) Group members involved in each project are listed at the end